Structural and Functional Studies of Ubiquitin Binding Domains The covalent modification of proteins by ubiquitination is a major regulatory mechanism of protein degradation and quality control, endocytosis, vesicular trafficking, cell-cycle control, stress response, DNA repair, growth factor signaling, transcription, gene silencing, and other areas of biology. A class of specific ubiquitin binding domains mediates most of the effects of protein ubiquitination. The known membership of this group has expanded rapidly and now includes at least sixteen domains. The structures of many of the complexes with monoubiquitin have been determined, revealing interactions with multiple surfaces on ubiquitin. Inroads into understanding polyubiquitin specificity have been made for two UBA domains, whose structures have been characterized in complex with Lys48-linked diubiquitin. Several ubiquitin binding domains, including the UIM, CUE, and A20 ZnF, promote autoubiquitination, which regulates the activity of proteins that contain them. At least one of these domains, the A20 ZnF, acts as a ubiquitin ligase by recruiting a ubiquitin:ubiquitin conjugating enzyme thiolester adduct in a process that depends on the ubiquitin-binding activity of the A20 ZnF. The affinities of the monoubiquitin binding interactions of these domains span a wide range, but are most commonly weak, with Kd >100 mM. The weak interactions between individual domains and monoubiquitin are leveraged into physiologically relevant high affinity interactions via several mechanisms: ubiquitin polymerization, modification multiplicity, oligomerization of ubiquitinated proteins and binding domain proteins, tandem binding domains, binding domains with multiple ubiquitin binding sites, and cooperativity between ubiquitin binding and binding through other domains to phospholipids and small G-proteins. The long terms goals of this project are to 1) determine the structural features of ubiquitin and its binding domains that are involved in molecular recognition;2) correlate structural features with functional properties of these proteins in trafficking;and 3) understand the mechanisms whereby low-affinity interactions between individual binding domains and ubiquitin moieties and leveraged into physiological recognition events. The main focus of this project in 2010 was to extend determine how the structure of ESCRT-0 influences its ability to bind a diverse set of ubiquitinated receptors. VHS (Vps27, Hrs, and STAM) domains occur in ESCRT-0 subunits Hrs and STAM, GGA adaptors, and other trafficking proteins. The structure of the STAM VHS domain-ubiquitin complex was solved at 2.6 resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS-domain containing proteins in yeast and humans, including both subunits of ESCRT-0, bound ubiquitin in vitro. ESCRTs have been implicated in sorting of Lys63-linked polyubiquitinated cargo. Intact human ESCRT-0 binds Lys63-linked tetraubiquitin 50-fold more tightly than monoubiquitin, though only two-fold more tightly than Lys48-linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly-connected VHS and UIM motifs of ESCRT-0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin binding sites in yeast ESCRT-0 show that cooperation between them is required for the sorting of the Lys63-linked polyubiquitinated cargo Cps1 to the vacuole.